Numerous studies have focused on cataloging the genome `parts list', including transcripts, transcription factor (TF) binding sites, and chromatin states. Transcriptional regulatory networks have been inferred based on these data, leading to models of what TFs are master regulators at or near the top of the regulatory hierarchy versus lineage- or condition-specific TFs, which are downstream of those regulators. However, none of these approaches directly identifies which factors engage inaccessible chromatin to initiate the transcriptional regulatory cascades. Pioneer factors serve as keys to chromatin accessibility for binding by the majority of TFs in a cell, by binding stably to nucleosomal DNA (`pioneer binding') and thus increase the DNA enzymatic accessibility of the chromatin (`pioneer activity'), allowing the sequential recruitment of other TFs on inactive chromatin. By priming cis regulatory elements for subsequent transcriptional regulatory activity, pioneer factors serve as `gatekeepers' to cellular differentiation. Despite their importance, little is known about pioneer factors, and only a handful have been characterized. A major hurdle in characterization of pioneer factors is the lack of a robust, high- throughput functional assay. In this project, we will develop a new technology, termed Pioneer Interactions On Nucleosomal Engineered ARrays (PIONEAR), for high-throughput characterization of pioneer binding. We will use PIONEAR assays to survey the pioneer interactions of dozens of human TFs. TFs identified by PIONEAR assays to exhibit pioneer binding will be evaluated in vivo to examine chromatin decompaction on a broader scale. Identification of TFs that exhibit pioneer activity may lead to breakthroughs in directed cellular differentiation and reprogramming and the development of improved therapies to target cancer stem cells.